炭疽杆菌保护性抗原的人源性单克隆抗体的初步研制
发布时间：2019-01-15 07:08
【摘要】： 炭疽是由炭疽芽孢杆菌(Bacillus anthracis)引起的一种急性、烈性人畜共患病,因其发病率高、潜伏期短、致死率高、炭疽芽孢对环境具有极强抵抗力等特点而使炭疽芽孢杆菌常被用做生物武器或生物恐怖战剂。青霉素等抗生素药物是治疗炭疽的传统方法,但其缺点显著。治疗性抗体(中和抗体)是感染后唯一有效的治疗药物,其中人源性中和抗体由于疗效肯定、特异性高、毒副作用小而被公认为最理想的抗体形式,因此研究开发治疗炭疽的人源性中和抗体药物具有重要意义。 根据炭疽毒素的致病机制,保护性抗原的第四结构域(简称PAD4)与细胞受体结合后介导了水肿因子和致死因子进入细胞内发挥毒素作用,PAD4在炭疽毒素致病中起着基础性的作用。因此本研究将炭疽毒素PAD4进行原核表达和纯化,以重组蛋白作为抗原表位,利用噬菌体抗体库技术制备抗炭疽杆菌PAD4抗体,为研制治疗炭疽的人源性中和抗体药物奠定基础。 本研究依照炭疽杆菌PAD4的氨基酸序列(NCBI中的P13423序列,140aa)和基因序列(420bp,GenBank Accession:AY428556)设计并优化其核苷酸序列,在化学合成12条寡核苷酸片段的基础上,利用重叠延伸多聚酶链式反应(SOE-PCR)合成PAD4基因,克隆入表达载体中实现了PAD4与辅助噬菌体M13KO7GⅢ蛋白N1结构域的可溶性融合表达和纯化。其次,以高纯度的融合蛋白为靶抗原从全合成人源性噬菌体抗体库(库容为5×109)中筛选具有特异性的人源性噬菌体单链抗体,实现人源性抗体基因在大肠杆菌中的可溶性表达并鉴定表达产物与PAD4的特异性结合活性。 本研究采用重叠延伸PCR反应合成了PAD4基因,实现了大肠杆菌的高水平可溶性表达,表达产物约占细菌总蛋白量的36%,经亲和层析纯化获得了纯度达到电泳级以上的重组蛋白。经过筛选获得了四株与炭疽毒素PAD4特异性结合的人源性单链抗体AP、APD17、APD22和APDC。 由于筛选的噬菌体抗体APDC轻链CDR3中含有琥珀突变密码子TAG,因此设计并合成了两条引物,进行重叠延伸PCR将TAG定点突变为CAG。将改造的噬菌体抗体APDC基因和其余三株噬菌体抗体AP、APD17、APD22基因分别克隆入表达载体PTIG-TRX-E中,最终实现了四株抗体在大肠杆菌中的可溶性表达,经初步鉴定大肠杆菌表达的四株抗体AP、APD17、APD22和APDC能与炭疽毒素PAD4特异性结合。
[Abstract]:Anthrax is an acute, acute zoonosis caused by Bacillus anthracis (Bacillus anthracis), because of its high incidence, short incubation period and high mortality. Because of its strong resistance to environment, Bacillus anthracis is often used as biological weapon or bioterrorist warfare agent. Antibiotic drugs such as penicillin are traditional methods for anthrax treatment, but their disadvantages are obvious. Therapeutic antibody (neutralizing antibody) is the only effective therapeutic drug after infection. Human neutralizing antibody is recognized as the most ideal form of antibody because of its positive curative effect, high specificity and little side effects. Therefore, it is of great significance to study and develop human neutralizing antibody drugs for anthrax treatment. According to the pathogenicity mechanism of anthrax toxin, the fourth domain of protective antigen (PAD4) binds to cell receptor and mediates the entry of edema factor and lethal factor into the cell to play the role of toxin. PAD4 plays a fundamental role in the pathogenesis of anthrax toxin. Therefore, the anthrax toxin PAD4 was expressed and purified in prokaryotic expression and purified, and the recombinant protein was used as antigen epitope to prepare PAD4 antibody against Bacillus anthracis by phage antibody library technology, which laid a foundation for the development of human neutralizing antibody drug for anthrax treatment. In this study, the nucleotide sequences were designed and optimized according to the amino acid sequence (P13423, 140aa) and gene sequence (420 BP PAD4 Accession:AY428556) of Bacillus anthracis PAD4, and 12 oligonucleotide fragments were chemically synthesized. PAD4 gene was synthesized by overlapping extension polymerase chain reaction (SOE-PCR) and cloned into expression vector. The soluble fusion expression and purification of PAD4 and N1 domain of auxiliary phage M13KO7G 鈪，
本文编号：2408960
[Abstract]:Anthrax is an acute, acute zoonosis caused by Bacillus anthracis (Bacillus anthracis), because of its high incidence, short incubation period and high mortality. Because of its strong resistance to environment, Bacillus anthracis is often used as biological weapon or bioterrorist warfare agent. Antibiotic drugs such as penicillin are traditional methods for anthrax treatment, but their disadvantages are obvious. Therapeutic antibody (neutralizing antibody) is the only effective therapeutic drug after infection. Human neutralizing antibody is recognized as the most ideal form of antibody because of its positive curative effect, high specificity and little side effects. Therefore, it is of great significance to study and develop human neutralizing antibody drugs for anthrax treatment. According to the pathogenicity mechanism of anthrax toxin, the fourth domain of protective antigen (PAD4) binds to cell receptor and mediates the entry of edema factor and lethal factor into the cell to play the role of toxin. PAD4 plays a fundamental role in the pathogenesis of anthrax toxin. Therefore, the anthrax toxin PAD4 was expressed and purified in prokaryotic expression and purified, and the recombinant protein was used as antigen epitope to prepare PAD4 antibody against Bacillus anthracis by phage antibody library technology, which laid a foundation for the development of human neutralizing antibody drug for anthrax treatment. In this study, the nucleotide sequences were designed and optimized according to the amino acid sequence (P13423, 140aa) and gene sequence (420 BP PAD4 Accession:AY428556) of Bacillus anthracis PAD4, and 12 oligonucleotide fragments were chemically synthesized. PAD4 gene was synthesized by overlapping extension polymerase chain reaction (SOE-PCR) and cloned into expression vector. The soluble fusion expression and purification of PAD4 and N1 domain of auxiliary phage M13KO7G 鈪，
本文编号：2408960
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2408960.html
最近更新
教材专著
